The TMT-labeled samples were mixed in an equal proportion, desalted and fractionated on an offline high-performance liquid chromatography (HPLC) apparatus (Agilent 1220) using basic pH reverse-phase liquid chromatography (pH 8.0, XBridge C18 column, 4.6 mm×25 cm, 3.5 μm particle size). The fractions were dried and resuspended in 5% formic acid and analyzed by acidic pH reverse phase LC-MS/MS analysis. The peptide samples were loaded on a nanoscale capillary reverse phase C18 column (New Objective, 75 μm ID×∼40 cm, 1.9 μm C18 resin from Dr Maisch, GmbH) by an HPLC system (Waters nanoAcquity) and eluted by a 150 min gradient. The eluted peptides were ionized by electrospray ionization and detected by an inline Orbitrap Fusion mass spectrometer (ThermoFisher Scientific). The mass spectrometer is operated in data-dependent mode with a survey scan in Orbitrap (60,000 resolution, 2×106 AGC target and 50 ms maximal ion time) and MS/MS high-resolution scans (60,000 resolution, 1×105 AGC target, ∼150 ms maximal ion time, 38 HCD normalized collision energy, 1 m/z isolation window and 20 s dynamic exclusion).
Xbridge c18 column
Manufactured by Agilent Technologies
Sourced in Germany, United States
The XBridge C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a spherical silica-based stationary phase with a C18 alkyl bonded ligand, providing efficient and robust chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
1200 hplc system, by Agilent Technologies (2 mentions)
Nanoacquity, by Waters Corporation (1 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
1200 series system, by Agilent Technologies (1 mentions)
Amazon sl ion trap mass spectrometer, by Bruker (1 mentions)
2400 chn elemental analyzer, by PerkinElmer (1 mentions)
Aviii 600, by Bruker (1 mentions)
Maxis uhr esi time of flight mass spectrometer, by Bruker (1 mentions)
Avance 3 500, by Bruker (1 mentions)
Milli q advantage, by Merck Group (1 mentions)
1260 infinity hplc, by Agilent Technologies (1 mentions)
6500 q tof, by Agilent Technologies (1 mentions)
1100 series, by Agilent Technologies (1 mentions)
High performance liquid chromatography system, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
9 protocols using xbridge c18 column
1
Offline HPLC Fractionation and LC-MS/MS Analysis
2
Synthesis and Characterization of K2[PtCl4]
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Potassium tetrachloridoplatinate (K2[PtCl4]) was purchased from Johnson Matthey (Switzerland). Water for synthesis was taken from a reverse osmosis system. For HPLC measurements Milli-Q water (18.2 MΩ cm, Merck Milli-Q Advantage, Darmstadt, Germany) was used. Other chemicals and solvents were purchased from commercial suppliers (Sigma Aldrich, Merck and Fisher Scientific). Electrospray ionization (ESI) mass spectra were recorded on a Bruker Amazon SL ion trap mass spectrometer in positive and/or negative mode by direct infusion. High resolution mass spectra were measured on a Bruker maXis™ UHR ESI time of flight mass spectrometer. One- and two-dimensional 1H-NMR and 13C-NMR spectra were recorded on a Bruker Avance III 500 or AV III 600 spectrometer at 298 K. For 1H-NMR spectra the solvent residual peak was taken as internal reference. Elemental analysis measurements were performed on a PerkinElmer 2400 CHN Elemental Analyzer at the Microanalytical Laboratory of the University of Vienna. The compounds were purified by preparative RP-HPLC using a Waters XBridge C18 column on an Agilent 1200 Series system. Milli-Q water and acetonitrile were used as eluents with a flow rate of 17 ml min−1, unless otherwise stated.
3
HPLC Analysis of Plant Extracts
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An Agilent 1100 liquid chromatography system equipped with an xBridge C18 column (250 mm, 4.6 mm, 5 µm) and UV detector set to three wavelengths (210, 254, and 360 nm) was used to analyze the contents in the plant extracts. The extracts were dried and weighed. Solutions with a concentration of 100 mg/L were prepared in a mixture of acetonitrile–water (1:1) and injected (10 µL or 100 µL) using an autosampler. Mobile phase A was acetic acid 0.1% in water. Mobile phase B was pure acetonitrile. The other separation conditions were a flow rate of 1 mL/min and a temperature of 30 °C.
4
Reversed-phase Fractionation of Peptides
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The peptides were further fractionated using a reversed-phase Waters XBridge C18 column (250 mm × 4.6 mm column packed with 3.5-μm particles) on an Agilent 1200 HPLC System (solvent A: 5 mM ammonium formate, pH 10, 2% ACN; solvent B: 5 mM ammonium formate, pH 10, 90% ACN) operating at a flow rate of 1 mL/min [Anal. Chem. 2019, 91, 9, 5794–5801]. Peptides were separated by a gradient mixture from 0% B to 16% B in six minutes, 40% B in 60 minutes, 44% B in 4 min and ramped to 60% B in five minutes. The 60% B mixture was kept for 14 min. Fractions were collected into a 96 well plate during the fractionation run with a total of 96 fractions at the 1-minute time interval. The 96 fractions were subsequently concatenated into 24 fractions by combining 4 fractions that are 24 fractions apart (i.e., combining fractions #1, #25, #49, and #73; #2, #26, #50, and #74; and so on). For proteome analysis, 5% of each concatenated fraction was dried down and re-suspended in 2% acetonitrile, 0.1% formic acid to a peptide concentration of 0.1 mg/mL for LC-MS/MS analysis. The rest of the fractions (95%) were further concatenated into 12 fractions (i.e., by combining fractions #1 and #13; #3 and #15; and so on), dried down, and phosphopeptides enriched using immobilized metal affinity chromatography (IMAC).
5
Encapsulin LC-MS Flavin Analysis
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Purified encapsulins were analyzed by LC–MS. The sample was buffer exchanged into water and then injected onto a Waters XBridge C18 column with an Agilent Infinity 1260 HPLC. The sample was eluted with a gradient of water to acetonitrile both supplemented with 0.1% formic acid. Mass analysis was performed with an Agilent 6500 Q-TOF operated in positive ion mode. The organic solvent denatures the encapsulin particles on the column thus releases the bound flavin species.
6
HPLC-MS Analysis of Thailandins
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The extract was analyzed by a HPLC system equipped with a photodiode array detector (200–600 nm) a mass spectrometer (1100 Series, Agilent Technologies, Waldbronn, Germany) The separation was done by usage of a XBridge™ C18 column (4.6 × 100 mm) with precolumn (4.6 × 20 mm) on a non-linear 0.5% AcOH-CH3CN:H2O gradient ranged from 20% to 95% at a flow rate of 0.5 mL/min. Thailandin A has UV/vis maxima at 325, 340, and 358 nm and a mass of 754 g/mol, thailandin B has 608 g/mol.
7
Potato pH and Organic Acid Analysis
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The pH of the potatoes was measured using a pH meter (Metler-Toledo Instruments Co., Ltd., Shanghai, China). A 2 g sample was placed in a centrifugal tube, to which 3 mL of ultra-pure water was then added. Then, it was centrifuged for 15 min at 10,000 rpm after ultrasonic treatment for 30 min in a water bath. The supernatants were filtered through a 0.22 μm polytetrafluoroethylene membrane before being injected. Organic acids (lactic acid and acetic acid) were analyzed using a high-performance liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump, an oven for controlling the column temperature (which was set at 25 °C), a UV-vis detector and a data acquisition system fitted with a XBridge® C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of a 0.02 M solution of potassium dihydrogen phosphate buffer (pH 2.88) and methanol (98:2, v/v). The autosampler was adjusted to an injection volume of 20 μL. Isocratic elution was applied to the mobile phase with a flow rate of 0.6 mL/min. A wavelength of 210 nm was selected for quantification. The calibration curves for each compound were constructed using pure standards at different concentrations [23 (link)].
8
Reversed-Phase Fractionation of Peptides
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The experiment was performed as described previously (35 (link)). In brief, the peptides were further fractionated using a reversed-phase Waters XBridge C18 column (250-mm × 4.6-mm column packed with 3.5 μmol/L particles) on Agilent 1200 HPLC System (solvent A: 5 mmol/L ammonium formate, pH 10, 2% ACN and solvent B: 5 mmol/L ammonium formate, pH 10, 90% ACN) operating at a flow rate of 1 mL/min. Peptides were separated by a gradient mixture from 0% B to 16% B in 6 minutes, 40% B in 60 minutes, 44% B in 4 minutes, and ramped to 60% B in 5 minutes. The 60% B mixture was kept for 14 minutes. Fractions were collected from at 2 to 95 minutes during the fractionation run, and a total of 96 fractions was collected at equal time intervals. These 96 fractions were subsequently concatenated into 12 fractions for global proteome and six fractions for phosphoproteome analysis. Each pooled fraction was dried down by vacuum centrifugation.
9
Fractionation and Phosphopeptide Enrichment
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